Reperfusion injury following hepatic ischemia is associated with inflammation and ongoing necrosis that is amplified by deletion of the A2A adenosine receptor (A2AR)1. The activity of most inflammatory cells, including but not limited to macrophages, monocytes, T lymphocytes, platelets and polymorphonuclear leukocytes, is inhibited by the activation of the anti-inflammatory Gs-coupled A2AR, resulting in reduced pro-inflammatory cytokine production and diminished endothelial adhesion molecule expression2-7. Accumulating evidence suggests that hepatic reperfusion injury is triggered by lymphocyte activation1 and that the activation of A2ARs on bone-marrow-derived cell mediates liver protection8. These findings, and studies establishing that the activation of the A2AR on CD4+ T cells inhibits TCR-mediated IFN-γ production in vitro3, suggest that treatment with the selective A2AR agonist, 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e), may mediate protection from hepatic ischemia reperfusion injury (IRI) by inhibiting the activation of CD4+ T lymphocytes. However, the rapidity of reperfusion injury is not consistent with the timeframe required for activation and differentiation of conventional CD4+ T cell responses, suggesting it is mediated by a rapidly activated T cell subset.
Most therapeutic studies involving CD1-restricted T cells have been confined to the use of a population of CD1d-restricted T cells, which expresses natural killer (NK) cell markers such as NK 1.1 and a T cell receptor (TCR) consisting of an invariant α-chain (Vα14Jα18 in mice and Vα24Jα18 in humans), which pairs with one of a limited number of β-chains. In both species, these “invariant” NKT cells display rapid and potent cytolytic activity and secretion of cytokines (IFN-γ, IL-2, IL-4 and IL-10), which direct adaptive immune responses.
The majority of mouse CD4+NK1.1+ natural killer T cells express the invariant TCR, Vα14Jα18, and are dependent on CD1d for positive selection in the thymus and subsequent activation in the periphery9;10. CD1d is expressed by hepatocytes, gut epithelial cells and APCs and presents either self glycolipid, such as isoglobotrihexosylceramide11, or foreign glycolipid, such as the marine sponge-derived α-Gal-Cer12, to NKT cells. The rapid release of IFN-γ or IL-4 following activation of invariant NKT (iNKT) cells by CD1d-glycolipid presentation to TCRs has been attributed to pre-formed cytokine transcripts13. Although NKT cells comprise only 0.1-3% of the T lymphocyte population in blood and spleen, in the murine liver NKT cells account for as much as 30% of the total lymphocyte population and as much as 50% of total αβ TCR+ T cells14. The high abundance of NKT cells in the liver and their rapid response to activation suggests that they might play a role in hepatic reperfusion injury. We show that NKT cells are involved in the pathogenesis of hepatic IRI and that they comprise a subset of CD4+ T lymphocytes through which ATL146e mediates liver protection.
While mice encode a single CD1 isoform, CD1d, humans have five isoforms, CD1a-e, of which CD1a, CD1b, CD1c and CD1d have been shown to stimulate T cells. CD1a, b and c are expressed by dendritic cells and other antigen-presenting cells and can present a range of lipids (mycolic acids, mycolates esterified to simple sugars, phosphatidyl inositol moieties linked to complex glycans, polyisoprenoid lipids and lipopeptides) derived from the cell walls of mycobacteria to T cells. CD1d is expressed by various myeloid, epithelial, parenchymal, and vascular smooth muscle cells in non-lymphoid organs and can present the α-anomeric glycolipid, α-galactosylceramide (αGC), found in marine sponges and glycosphingolipids from Gram-negative bacteria that do not contain lipopolysaccharide to subsets of T cells. Many CD1-restricted T cells can also be stimulated by exposure to antigen-presenting cells expressing CD1a, CD1b, CD1c or CD1d in the absence of added antigen. This autoreactivity probably results from T cell recognition of endogenous lipid antigens, such as phospholipids, gangliosides, sulphatides and the lysosomal glycosphingolipid, isoglobotrihexosylceramide, which have been shown to bind to human and murine CD1 and activate CD1-restricted T cells.